Scytophycins-producing Scytonema pseudohofmanni microorganisms

ABSTRACT

Scytophycins A, B, C, D and E, an aldehyde and alcohol derivative of scytophycin B and the specified alkanoyl and benzoyl esters of scytophycins A, B, C, D and E and the B derivatives (scytophycin compounds) are novel antineoplastic and antifungal agents. Antifungal compositions containing a scytophycin compound and methods of inhibiting fungi, especially plant pathogenic fungi, using these compositions are included. A method for producing the scytophycin complex, comprising scytophycins A, B, C, D and E, using a new strain of the blue-green alga Scytonema pseudohofmanni, and a biologically purified culture of the alga are also provided.

This invention was supported in part by a grant (PHS grant CA12623) ofthe National Cancer Institute, Department of Health and Human Services.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 07/401,353, filedAug. 31, 1989, U.S. Pat. No. 4,996,229 which in turn is a division ofapplication Ser. No. 06/865,499, filed May 21, 1986, U.S. Pat. No.4,863,955 which is a continuation-in-part of copending applications Ser.Nos. 741,776 and 741,778, both filed Jun. 6, 1985 both now abandoned.

SUMMARY OF THE INVENTION

This invention relates to five new pharmacologically active compounds,scytophycins A, B, C, D and E, which have been isolated from theblue-green alga Scytonema pseudohofmanni, to an aldehyde and alcoholderivative of scytophycin B and to certain acyl esters of thesecompounds. In other aspects, this invention relates to a method forproducing scytophycins A, B, C, D and E by culturing a new strain ofScytonema pseudohofmanni, Bharadwaja ATCC 53141, and to a biologicallypurified culture of the novel S. pseudohofmanni strain.

This invention further relates to a method for using scytophycins A, B,C, D and E and specified acyl esters of these compounds to inhibitfungi, in particular, fungal plant pathogens. Fungicidal compositionscontaining scytophycins A, B, C, D and E and their specified esters arealso part of this invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the mature filaments of the new strain of Scytonemapseudohofmanni Bharadwaja (Nostocales, Scytonemataceae). As shown, thefilaments have single and geminate false branches and single intercalaryheterocysts somewhat broader than the trichome. The scale bar represents20 μm.

FIG. 2 shows the 360 MHz proton nuclear magnetic resonance (¹ H NMR)spectrum of scytophycin A in acetone d₆.

FIG. 3 shows the 360 MHz ¹ H NMR spectrum of scytophycin B in acetoned₆.

DETAILED DESCRIPTION OF THE INVENTION

Neoplastic diseases (cancer) continue to be a widespread problem.Although the mechanisms by which such diseases invade the body have beenunder intensive investigation for a number of years, much remains to belearned before this problem can be solved. Thus, novel antineoplasticagents are quite useful because they contribute to a generalunderstanding of the underlying mechanisms by which malignant cellsproliferate.

Fungal plant pathogens cause great economic losses each year. New agentseffective against these phytopathogens are needed. Presently usedfungicides differ in their effectiveness against specific fungi. Inaddition, resistant fungal strains frequently develop, creating acontinual need for new, effective agents. Furthermore, some fungicidesare difficult to obtain and, thus, expensive to use. Fungicides whichare less expensive are, therefore, quite beneficial.

This invention relates to a compound selected from scytophycins A, B, C,D and E, an aldehyde and primary alcohol derivative of B, and the C₁ -C₆-alkanoyl and benzoyl and the halo-, hydroxy- and C₁ -C₃-alkoxy-substituted C₁ -C₆ -alkanoyl and benzoyl esters of scytophycinsA, B, C, D and E and the specified B derivatives. For convenience, thesecompounds will be called scytophycin compounds.

The scytophycin compounds are potent cytotoxins and antineoplasticagents. In addition, they inhibit fungal pathogens. For example,scytophycins A and B inhibit Candida albicans, the causative agent forcandidiasis in animals and man. The scytophycin compounds also inhibitfungal plant pathogens such as Pythium ultimum (Phycomycetes), whichcauses damping-off diseases of seedlings, Rhizoctonia solani(Basidiomycetes), which causes damping-off and stem rot and Sclerotiniahomoeocarpa (ascomycetes), which causes diseases of turf grasses.

Thus, this invention further relates to a method for treating pathogenicfungi which comprises contacting the loci of the fungi with afungicidally-effective amount of a scytophycin compound.

The scytophycin complex, comprising major factors A, B and C and minorfactors D and E, was isolated from a strain of an epidaphic variety ofthe blue-green alga Scytonema pseudohofmanni Bharadwaja(Scytonemataceae). The new strain is a clonal culture isolated afterrepeated subculture of an algal sample collected in a moist, heavilyforested region of the Koolauloa District on the island of Oahu, Hi.

The Scytonema pseudohofmanni culture of this invention has beendeposited and made part of the stock culture collection of the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852,from which it is available to the public under the accession number ATCC53141.

As is the case with other organisms, the characteristics of Scytonemapseudohofmanni ATCC 53141 are subject to variation. For example,recombinants, variants or mutants of the ATCC 53141 strain may beobtained by treatment with various known physical and chemical mutagens,such as ultraviolet rays, X rays, gamma rays, andN-methyl-N'-nitro-N-nitrosoguanidine. All natural and induced variants,mutants and recombinants of Scytonema pseudohofmanni ATCC 53141 whichretain the characteristic of producing scytophycin A, B, C, D or E maybe used in this invention.

Scytophycin B, which is the major metabolite produced by the newScytonema pseudohofmanni strain, is believed to have the structure shownin formula 1. ##STR1## Scytophycin B has the following characteristics:

Form: white amorphous solid.

Empirical formula: C₄₅ H₇₃ NO₁₂.

Molecular weight: 819 [determined by fast-atom bombardment massspectrometry (FABMS)].

UV λ_(max) (ethanol): 264 nm (ε28,500).

[α]_(D) ²⁰ : -24° (c 0.63, MeOH).

CD (MeOH): [θ]₂₆₆ +16,000.

IR (CHCl₃): 1690, 1660 and 1125 cm⁻¹.

¹ H NMR: (Table 1)

¹³ C NMR: (Table 3)

MS (FAB): m/z 858 (M+K)⁺, 842 (M+Na)⁺ and 820 (M+H)⁺ ; Found m/z802.5123. Calcd for C₄₅ H₇₂ NO₁₁ : (M-H₂ O+H) 802.5105.

Scytophycin A, which is a second metabolite produced by the S.pseudohofmanni culture, is believed to have the structure shown informula 2. ##STR2## Scytophycin A has the following characteristics:

Empirical formula: C₄₅ H₇₅ NO₁₂.

Molecular weight: 821 (determined by FABMS).

UV λ_(max) (ethanol): 264 nm (ε35,000).

Form: white amorphous solid.

[α]_(D) ²² : -35° (c 1.3, MeOH)

CD (MeOH): [θ]₂₆₆ +12,200

IR (CHCl₁₃): 1690, 1660 and 1120 cm⁻¹

¹ H NMR: (Table 1)

¹³ C NMR: (Table 3)

MS (FAB): m/z 860 (M+K)⁺, 844 (M+Na)⁺ and 822 (M+H)⁺ ; Found m/z822.5407. Calcd for C₄₅ H₇₆ NO₁₂ : (M+H) 822.5368.

Table 1 summarizes and compares the ¹ H NMR spectral data forscytophycins A and B. The spectra are shown in FIGS. 2 and 3.

                  TABLE 1                                                         ______________________________________                                        The .sup.1 H NMR Data in Acetone d.sub.6 for                                  Scytophycins A and B.                                                         Assignment   Scytophycin B                                                                             Scytophycin A                                        ______________________________________                                         1           --          --                                                    2           5.78        5.78                                                  3           7.66        7.65                                                  4           --          --                                                    4-Me        1.85        1.80                                                  5           6.02        6.01                                                  6           2.48        2.43                                                              2.57        2.57                                                  7           4.06        4.06                                                  8           1.26        ˜1.2                                                        1.77        1.71                                                  9           4.58        4.57                                                 10           5.66        5.66                                                 11           5.81        5.81                                                 12           1.91        1.91                                                 13           3.39        3.38                                                 14           1.46        1.46                                                              1.55        1.54                                                 15           3.94        3.93                                                 15-OMe       3.37        3.37                                                 16           --          --                                                   CH.sub.2 -16 2.63        2.63                                                              2.72        2.72                                                 17           3.88        3.84                                                 17-OMe       3.24        3.24                                                 18           1.46        1.43                                                              1.96        1.94                                                 19           3.31        3.30                                                 19-OMe       3.20        3.19                                                 20           2.09        2.09                                                 20-Me        0.86        0.84                                                 21           5.22        5.20                                                 22           1.99        1.93                                                 22-Me        0.88        0.84                                                 23           3.03        3.02                                                 24           1.69        1.68                                                 24-Me        0.99        1.00                                                 25           1.37         NA*                                                              1.75        NA                                                   26           2.54        2.29                                                                          1.55                                                 27           --          3.39                                                 28           2.78        1.58                                                              (2.74)**                                                         28-Me        0.92        0.84                                                 29           3.28        3.12                                                 29-OMe       3.31        3.12                                                 30           2.46        2.51                                                 30-Me        1.15        1.13                                                 31           5.12        5.11                                                              (5.18)      (5.19)                                               32           6.79        6.74                                                              (7.11)      (7.09)                                               33-NMe       2.99        2.96                                                              (3.11)      (3.07)                                               34           8.36        8.35                                                              (8.11)      (8.09)                                               ______________________________________                                         *NA = not assigned                                                            **() indicates chemical shift of protons in minor conformer due to            restricted rotation around C(32)--N(33) and N(33)--C(34) bonds           

Scytophycin C, a third metabolite produced by the S. pseudohofmanniculture, is believed to have the structure shown in formula 3 ##STR3##Scytophycin C has the following characteristics:

Form: white amorphous solid.

Empirical Formula: C₄₅ H₇₅ NO₁₁.

Molecular Weight: 805.

[α]_(D) ¹⁸ : -23° (c 1.2, MeOH).

CD (MeOH): [θ]₂₆₅ +10,600.

UV λ_(max) (MeOH): 262 nm (ε48,000).

IR (CHCl₃): 1690, 1660 and 1115 cm⁻¹.

¹ H NMR: (Table 2).

¹³ C NMR: (Table 3).

MS (FAB): m/z 828 (M+Na)⁺, 788 (M--H₂ O+H)⁺ and 156 (CH₃ O⁺═CH--CH(CH₃)--CH═CH--N(CH₃)--CHO).

Scytophycin D, which is a minor metabolite produced by the S.pseudohofmanni culture, is believed to have the structure shown informula 4. ##STR4## Scytophycin D has the following characteristics:

Form: white amorphous solid.

Empirical Formula: C₄₅ H₇₅ NO₁₂.

Molecular Weight: 821.

[α]_(D) ²⁰ : -32° (c 0.90, MeOH).

CD (MeOH): [θ]₂₆₅ +13,400.

UV λ_(max) (MeOH): 264 nm (ε47,000).

IR (CHCl₃): 1690, 1660 and 1120 cm⁻¹.

¹ H NMR: (Table 2).

¹³ C NMR: (Table 3).

MS (FAB): m/z 844 (M+Na)⁺, 804 (M--H₂ O+H)⁺ and 156 (CH₃ O⁺═CH--CH(CH₃)--CH═CH--N(CH₃)--CHO).

Scytophycin E, which is another minor metabolite produced by the S.pseudohofmanni culture, is believed to have the structure shown informula 5. ##STR5## Scytophycin E has the following characteristics:

Form: white amorphous solid.

Empirical Formula: C₄₅ H₇₅ NO₁₂.

Molecular Weight: 821.

[α]_(D) ¹⁹ : -38° (c 1.0, MeOH).

CD (MeOH): [θ]₂₆ +27,000.

UV λ_(max) (MeOH): 265 nm (ε39,000).

IR (CHCl₃): 1690, 1660 and 1090 cm⁻¹.

¹ H NMR: (Table 2).

¹³ C NMR: (Table 3).

MS (FAB): m/z 844 (M+Na)⁺, 804 (M--H₂ O+H)⁺ and 156 (CH₃ O⁺═CH--CH(CH₃)--CH═CH--N(CH₃)--CHO); Found: m/z 844.5190. Calcd for C₄₅H₇₅ NO₁₂ Na: (M+Na)⁺ 844.5187.

The scytophycin factors thus appear to have the common structure shownin formula 6.

    __________________________________________________________________________     ##STR6##                                             6                       No.                 Compound                                                  __________________________________________________________________________    1                   Scytophycin B: R.sup.1 and R.sup.2 = O; R.sup.3 and                           R.sup.4 = O                                               2                   Scytophycin A: R.sup.1 and R.sup.2 = O; R.sup.3 = H;                          R.sup.4 = OH                                              3                   Scytophycin C: R.sup.1 = R.sup.2 = H; R.sup.3 and                             R.sup.4 = O                                               4                   Scytophycin D: R.sup.1 = OH; R.sup.2 = H; R.sup.3 and                         R.sup.4 = O                                               5                   Scytophycin E: R.sup.1 = H; R.sup.2 = OH; R.sup.3 and                         R.sup.4 = O                                               __________________________________________________________________________

Table 2 summarizes and compares the ¹ H NMR spectral data forscytophycins C, D and E.

                  TABLE 2                                                         ______________________________________                                        .sup.1 H NMR Data for Scytophycins C, D and E                                 in Acetone- -d.sub.6 (δ)                                                       Scytophycin                                                            Assignment                                                                             C           D           E                                            ______________________________________                                         2       5.78 d (15.8)                                                                             5.76 d (15.6)                                                                             5.75 d (15.6)                                 3       7.61 d (15.8)                                                                             7.47 d (15.6)                                                                             7.64 d (15.6)                                Me on 4  1.82 br s   1.84 br s   1.82 br s                                     5       6.03 br d   5.87 br t   6.01 br d                                             (9, 4.5)    (8)         (8.7, 3.9)                                    6       2.46        2.50        2.52                                          6'      2.46                                                                  7       4.02 t      4.01 br s   4.03 br t                                     8       1.28        1.22        1.34                                          8'      1.77        1.73        1.78                                          9       4.53 br d (9)                                                                             4.47 br d (10.5)                                                                          4.57 br d (7.5)                              10       5.67 br d (10)                                                                            5.63 br d (10)                                                                            5.66 br d (10.5)                             11       5.77 m      5.76 m      5.78 m                                       12       1.89        2.05        1.95                                         12'      *           *           *                                            13       3.34        3.27        3.28                                         14       1.68        1.85        1.86                                         14'      1.63        1.60        1.79                                         15       3.62 br d   3.58 br s   3.80                                         MeO on 15                                                                              3.30 s      3.38 s      3.36 s                                       16       1.68                    1.67                                         Me on 16 0.80 d (6.9)                                                                              1.00 s                                                   CH.sub.2 on 16                   3.80 br d (9)                                                                 3.65 m                                       17       3.49 dd     3.52 dd     3.47 t                                       MeO on 17                                                                              3.23 s      3.24 s      3.18 s                                       18       1.78        1.97        1.97                                         19       3.47        3.45        3.74                                         MeO on 19                                                                              3.17 s      3.39 s      3.19 s                                       20       2.04        2.16        2.10                                         Me on 20 0.89 d (7.1)                                                                              0.87 d (7.6)                                                                              0.90 d (6.9)                                 21       5.16 br d (10.5)                                                                          5.27 br d (10)                                                                            5.18 br d (9.6)                              22       2.00        1.96        2.03                                         Me on 22 0.84 d (6.9)                                                                              0.84 d (6.6)                                                                              0.85 d (6.9)                                 23       3.00        3.01        3.13                                         OH on 23 *           3.88 d (4.5)                                                                              4.12 d (4.5)                                 24       1.67        1.66        1.74                                         Me on 24 0.97 d (6.7)                                                                              0.95 d (6.7)                                                                              0.97 d (6.6)                                 25       1.38        1.42        1.35                                         25'      1.76        1.74        1.71                                         26       2.55        2.53        2.54                                         26'      2.55        2.53        2.54                                         28       2.77        2.76        2.76                                         Me on 28 0.90 d (7.0)                                                                              0.90 d (7.1)                                                                              0.90 d (6.9)                                 29       3.27        3.30        3.28                                         MeO on 29                                                                              3.29 s      3.29 s      3.30 s                                       30       2.44        2.41        2.47                                         Me on 30 1.13 d (7.0)                                                                              1.13 d (6.9)                                                                              1.13 d (6.9)                                 31       5.12 dd     5.10 dd     5.10 dd                                               (14.2, 9.3) (14.1, 9)   (14.1, 9)                                    31**     5.17 dd     5.16 dd     5.17 dd                                               (14.7, 9)   (14.7, 9)   (14.7, 9)                                    32       6.77 d (14.2)                                                                             6.77 d (14.1)                                                                             6.77 d (14.1)                                32**     7.09 d (14.7)                                                                             7.09 d (14.7)                                                                             7.09 d (14.7)                                Me on N-33                                                                             2.97 s      2.97 s      2.97 s                                       Me on    3.09 s      3.09 s      3.09 s                                       N-33**                                                                        34       8.34 s      8.34 s      8.34 s                                       34**     8.09 s      8.10 s      8.10 s                                       ______________________________________                                         *not assigned                                                                 **signals of the minor conformer                                         

Table 3 summarizes the ¹³ C NMR chemical shift data for scytophycins A,B, C, D and E.

                  TABLE 3                                                         ______________________________________                                        .sup.13 C NHR Data for Scytophycins A, B, C, D                                and E in Acetone- -d.sub.6                                                            Scytophycin                                                           Assignment.sup.a                                                                        A       B.sup.b  C      D      E                                    ______________________________________                                         1        169.57  169.63   169.38 168.83 169.61                                2        115.63  115.58   115.71 116.38 115.68                                3        151.81  151.85   151.33 151.12 151.70                                4        134.77  134.76   134.69 135.03 134.76                               Me on 4   12.30   12.29    12.09  12.27  12.24                                 5        139.90  139.95   139.73 139.55 139.93                                6        41.54   42.08    41.92  42.10  42.11                                 7        68.91   68.86    68.54  68.02  68.82                                 8        41.06   41.51    41.19  40.48  41.61                                 9        70.79   70.91    70.76  70.39  70.98                                10        131.52  131.51   131.48 131.32 131.67                               11        125.02  124.90   124.48 124.85 124.76                               12        31.85   31.84    32.19  31.18  31.48                                13        66.86   66.87    65.75  65.94  66.04                                14        34.76   35.63    32.61  32.50  32.21                                15        78.42   78.44    79.79  78.46  79.94                                MeO on 15 57.46   57.46    56.46  57.19  57.12                                16        61.69   61.12    40.93  82.73  46.95                                CH.sub.2 (.sub.3)-16                                                                    45.66   45.56    9.25   18.08  59.58                                17        75.27   75.20    76.32  76.25  76.70                                MeO on 17 52.89   52.83    53.55  58.25  51.87                                18        27.49   27.45    27.31  27.92  27.18                                19        77.47   77.49    77.93  77.35  77.44                                MeO on 19 57.78   57.78    58.03  58.25  58.12                                20        38.14   38.15    40.29  40.36  40.02                                Me on 20  9.36    9.23     9.05   9.23   9.37                                 21        76.51   76.51    76.56  76.52  76.52                                22        37.90   38.15    38.45  38.19  38.80                                Me on 22  9.27    9.23     8.79   9.23   9.27                                 23        76.51   76.51    77.18  76.44  76.30                                24        34.29   33.78    33.69  33.87  33.85                                Me on 24  18.63   18.27    18.13  18.25  18.31                                25        25.74   22.71    22.55  22.74  22.71                                26        39.39   39.36    39.17  39.48  39.43                                27        71.00   214.07   213.84 213.98 213.93                               28        35.72   49.48    49.33  49.50  49.50                                Me on 28  9.84    13.65    13.47  13.65  13.64                                29        88.66   88.26    88.11  88.28  88.27                                MeO on 29 61.13   61.12    60.96  61.13  61.14                                30.sup.c  38.53   37.98    38.03  36.83  38.21                                30        38.71   38.35    38.18  38.36  38.36                                Me on 30  20.06   19.62    19.45  19.62  19.63                                31        112.27  111.18   110.97 111.10 111.09                               31.sup.c  114.20  113.25   113.05 113.18 113.16                               32        129.65  130.15   129.98 130.17 130.17                               32.sup.c  124.76  125.44   125.46 125.46 125.45                               Me on N-33                                                                              27.22   27.22    27.03  27.19  27.18                                Me on N-33.sup.c                                                                        33.02   33.03    32.85  33.00  33.01                                34        162.81  162.89   162.71 162.83 162.84                               34.sup.c  161.52  161.60   161.62 161.58 161.58                               ______________________________________                                         .sup.a 75 MHz; acetoned.sub.6 as internal reference = 29.80 ppm.              .sup.b H-.sup.13 C connectivities determined using a phasecycled 16step       heteronuclear chemical shift correlation map experiment.                      .sup.c Signals for the minor conformer.                                  

Scytophycin B has been converted to two useful derivatives. Whenscytophycin B was treated with acetic acid and ethanol at roomtemperature for a day, the aldehyde of formula 7 was formed. Thisaldehyde was reduced with sodium borohydride to give the primary alcoholof formula 8. ##STR7##

The ¹ H NMR spectra of compounds 7 and 8 are summarized in Table 4.

                  TABLE 4                                                         ______________________________________                                        .sup.1 H NMR Spectra of Compounds  -7                                         and  -8 in Acetone- -d.sub.6 (δ)                                        Position.sup.a    -7           -8                                             ______________________________________                                        2                5.71         5.70                                            3                7.43         7.42                                            Me on 4          1.79         1.80                                            5                5.86         5.88                                            6                2.42                                                         6'               2.51                                                         7                3.96         3.97                                            8                1.25                                                         8'               1.78                                                         9                4.51         4.50                                            10               5.62         5.64                                            11               5.76         5.77                                            12               1.88                                                         13               3.40                                                         14               1.44                                                         14'              1.55                                                         15               3.84 dd      3.81                                            CH.sub.2 on 16   2.65 d                                                                        2.55 d                                                       17               3.77 dd                                                      18               1.44                                                         18'              1.90                                                         19               3.23                                                         20               1.98                                                         Me on 20         0.85         0.89                                            21               5.20         5.20                                            22               2.13                                                         Me on 22         0.77         0.78                                            23               3.08                                                         24               2.13                                                         Me on 24         1.06         1.06                                            25ax             1.12b                                                        25eq             1.25b                                                        26ax             0.90b                                                        25eq             1.77b                                                        28               1.73                                                         Me on 28         0.87         1.04                                            29               3.37                                                         30               2.31                                                         Me on 30         0.92         0.91                                            31               2.58                                                         32               9.81         3.64                                            32'              3.54                                                         OMe              3.36         3.38                                            OMe              3.31         3.27                                            OMe              3.19         3.19                                            OMe              3.19         3.19                                            ______________________________________                                         .sup.a 300 MHz; residual acetoned.sub.5 as internal reference = 2.04 ppm.     .sup.b Tentative assignments.                                            

As will be apparent from their structures, scytophycins A, B, C, D and Eand the derivatives of B have hydroxyl groups which are capable ofesterification. In addition, the primary alcohol of Compound 8 can beacylated. In scytophycin B, the hydroxyl group at C-7 is acylated morereadily than that at C-23. The C₁ -C₆ -alkanoyl and benzoyl esters orthe halo-, hydroxy-or C₁ -C₃ -alkoxy-substituted C₁ -C₆ alkanoyl orbenzoyl esters of the scytophycin factors and of compounds 7 and 8 arealso part of this invention.

At sublethal doses, the scytophycin compounds are antineoplastic agents.The minimum lethal dose of scytophycin B in mice, when administered IP,is about 650 μg/kg. Its toxicity is comparable to that of curare andstrychnine, but is almost two orders of magnitude less than that ofsaxitoxin and tetrodotoxin.

The scytophycin compounds are potent cytotoxins. For example, theminimum toxic doses of scytophycins A and B against KB human epidermoidcarcinoma and NIH/3T3 mouse fibroblast cell lines were determined to be1 ng/mL and 0.65 ng/mL, respectively, using methods described by E.Furusawa and W. Cutting in Ann. New York Acad. Sci. 173, 669-679 (1970).

Both scytophycin A and scytophycin B exhibited moderate activity againstintraperitoneally implanted P388 lymphocytic leukemia and Lewis lungcarcinoma, but did not exhibit activity against intraperitoneallyimplanted B16 melanoma.

Female hybrid BDF1 (DBA/2×C57BL/6) mice were used for the evaluation ofthe scytophycins against the three tumors. The mice were firstinoculated intraperitoneally with 10⁶ cells of P388 ascites leukemia(P388), 2 to 6×10⁵ cells of Lewis lung carcinoma (LLC) homogenate, or0.3 mL of 20% homogenate of B16 tumor (B16) masses. Drug treatment wasstarted the next day (day 1) and continued daily for 6-9 days. Theresults of these tests are summarized in Table 5.

                                      TABLE 5                                     __________________________________________________________________________    Effect of the Scytophycins on Intraperitoneally                               Implanted P388 Lymphocytic Leukemia, Lewis Lung                               Carcinoma, and B16 Melanoma in BDF1 Mice                                                            Mean Survival                                           Scyto-   Dose  Days of                                                                              Time, Days                                              phycin                                                                            Tumor                                                                              μg/Mouse                                                                         Treatment                                                                            Treated                                                                             Controls                                                                            % T/C.sup.a                                 __________________________________________________________________________    A   P388 1     8      10.0  8.0   125                                         B   P388 4     8      12.0  9.4   128                                                  3.25  6      11.4  8.4   136                                                  2     8      12.2  9.4   130                                                  1     8      10.6  9.4   113                                                  0.65  8      10.0  8.0   125                                                  0.5   8      9.4   9.4   100                                         B   LLC  4     9      12.6  9.8   129                                                  2     9      12.2  9.8   124                                                  1     9      11.2  9.8   114                                                  0.5   9      10.4  9.8   106                                         B   B16  8     8      12.0  19.0   63                                                                           (toxic)                                              4     8      19.8  19.0  104                                                  2     8      19.4  19.0  102                                         __________________________________________________________________________     .sup.a T/C = survival time of treated/survival time for control          

The scytophycins are also useful as antifungal agents. Thus, in oneaspect, this invention relates to a method of protecting plants fromphytopathogenic fungi which comprises contacting the loci of the fungiwith a fungicidally-effective amount of a scytophycin compound. The lociof the fungi can be a portion of the plant, such as leaves, stems,flowers or roots, or the soil wherein the fungi may be located.

Application rates will vary according to a number of factors, such asthe location of the plants being protected and the severity of thefungal infection. Thus, for use in a greenhouse, the fungicidal compoundis applied as a soil drench using a composition having a concentrationin the range of from about 1 to about 200 ppm of active ingredient,preferably from about 5 to about 100 ppm. As is understood by those inthe art, application rates used in the field are usually greater thanthose used in a greenhouse, and range from about 25 to about 1000 ppm.

In another embodiment, this invention relates to compositions suitablefor inhibiting plant-pathogenic fungi comprising 1) a scytophycincompound in an amount effective to inhibit the growth of aplant-pathogenic fungus and 2) a suitable carrier.

The compositions for use in this embodiment desirably contain, inaddition to the scytophycin compound, one or more of a number ofsuitable formulating carriers, including water, polyhydroxy compounds,petroleum distillates, and other dispersion media, surface-activedispersing agents, emulsifiers, and finely-divided inert solids. Theconcentration of scytophycin compound in these compositions will vary,depending on whether the composition is intended for direct applicationto plants or is intended to be subsequently diluted with an additionalinert carrier or carriers, such as water, to produce the ultimatetreating composition.

Treating compositions are most conveniently formulated by preparingliquid or solid concentrates, which are subsequently diluted to thedesired level for use. Emulsifiable liquid concentrates can be preparedby incorporating from about 1 to about 10 percent by weight of theactive ingredient and an emulsifiable agent in a suitablewater-immiscible organic liquid. Such concentrates may be furtherdiluted with water to form spray mixtures in the form of oil-in-wateremulsions. Such spray compositions then comprise active compound,water-immiscible solvent, emulsifying agent, and water. Suitableemulsifying agents can be of the nonionic or ionic types, or blendsthereof, and include condensation products of alkylene oxides withphenols and organic acids, polyoxyethylene derivatives of sorbitanesters, complex ether alcohols, ionics of the arylalkyl sulfonate type,and the like. Suitable water-immiscible organic liquids include aromatichydrocarbons, aliphatic hydrocarbons, cycloaliphatic hydrocarbons, andmixtures thereof such as petroleum distillates.

Solid concentrate mixtures can be prepared by incorporating from about10 to about 50% by weight of active compound in a finely-divided solidcarrier such as bentonite, Fuller's earth, diatomaceous earth, hydratedsilica, diatomaceous silica, expanded mica, talc, chalk, and the like.Such concentrates can be formulated, if desired, for direct use asdusting compositions, or can be diluted, if desired, with additionalinert solid carriers to produce dusting powders containing around 0.05to 1% by weight of a scytophycin compound. Alternatively, thesurfactants, that is, dispersing and/or wetting agents, can beincorporated along with the scytophycin compound in the solid carrier toform wettable powder concentrates ranging from about 10 to about 25% byweight concentration, which subsequently can be dispersed in water orother hydroxylated carrier to form spray compositions. Suitablesurfactants include condensed aryl sulfonic acids and sodium saltsthereof, sodium lignosulfate, sulfonate-oxide condensate blends,alkylaryl polyether alcohols, sulfonate/nonionic blends, anionic wettingagents, and the like.

Further, the scytophycin compound can be incorporated in solutions,simple dispersions, aerosol formulations, and other media acceptable fortreating vegetation or for applying to the soil.

The antifungal compositions of this embodiment are applied to infectedor susceptible plant or soil surfaces in any convenient fashion, such asby spraying, dusting, dipping, or drenching. A spray method isconsidered preferable, especially when large numbers of plants areinvolved, because such a treatment is faster and more uniform. Inspraying it is usually sufficient for the infected or susceptiblesurfaces to be made thoroughly wet with the liquid dispersion. Goodresults can be obtained by using spray compositions whether they areemulsions or aqueous dispersions of solid concentrates.

Where the fungi to be controlled are in the soil, the antifungalcompound can be applied to the soil directly, or it can be diluted withvarious inert solid or liquid diluents and then applied to the soil. Inone method of application the soil surface is sprayed with a liquiddispersion or emulsion of the active ingredient. The application isallowed to remain as a coating on the surface of the soil or,alternatively, is incorporated into the soil by disking, hoeing, orother methods known to those in the art. Another method of applicationis to apply the active ingredient in the form of a liquid dispersion oremulsion to the soil as a drench. Thus, for the control ofsoil-inhabiting fungi in the greenhouse, the application rate variesfrom about 5 to about 200 ppm active ingredient.

Scytophycin compounds can also be used as a seed soak for seeds prior toplanting. A suitable seed-soak formulation contains a scytophycincompound together with excipients such as a mixture of ethanol-acetone,polyoxyethylene sorbitan monolaurate, and the like.

When used as a seed soak, control can be accomplished at an applicationrate of from about 50 to about 400 ppm of scytophycin compound. Theseeds are allowed to soak in the formulation for about 4 hours and thenare removed and planted.

The activity of scytophycin compounds against pathogenic fungi instandard in vitro agar-diffusion tests, using 1/4" discs holding ˜30 μLof test solution, is illustrated in Tables 6-12.

                  TABLE 6                                                         ______________________________________                                        In Vitro Agar Diffusion Activity of                                           Scytophycins A and B vs. Pythium                                              ultimum X751                                                                  Compound     Level    Zone of Inhibition Size                                 ______________________________________                                        Scytophycin A                                                                              1 mg/mL  37 mm                                                   Scytophycin B                                                                              1 mg/mL  37 mm                                                   ______________________________________                                    

                  Table 7                                                         ______________________________________                                        In Vitro Agar Diffusion Activity of Scytophycins A                            and B vs. Fungal Pathogens                                                                Size of Zone of Inhibition (mm)                                           Concen-   Saccharomyces                                                                             Neurospora                                                                            Candida                                         tration   pastorianus crassa  albicans                                Compound                                                                              mg/mL     X52         X846    X657                                    ______________________________________                                        Scyto-  1         24          30      23                                      phycin A                                                                              0.1       17          25      19                                              0.01      T.sup.a     18      12                                      Scyto-  1         27          32      26                                      phycin B                                                                              0.1       20          27      22                                              0.01      20          30      21                                      ______________________________________                                         .sup.a T = trace                                                         

                  TABLE 8                                                         ______________________________________                                        In Vitro Agar Diffusion Activity of                                           Scytophycins B, D, and E vs. Fungal Pathogens                                             Size of Zone of Inhibition (mm)                                           Concen-   Saccharomyces                                                                             Neurospora                                                                            Candida                                         tration   pastorianus crassa  albicans                                Compound                                                                              mg/mL     X52         X846    X657                                    ______________________________________                                        Scyto-  1         24          36      31                                      phycin B                                                                              0.33      24          36      25                                      Scyto-  1         12          25      19                                      phycin D                                                                              0.33      T.sup.a     20      14                                      Scyto-  1         23          36      21                                      phycin E                                                                              0.33      18          26      22                                      ______________________________________                                         .sup.a T = trace                                                         

                  TABLE 9                                                         ______________________________________                                        In Vitro Agar Diffusion Activity of Scytophycin C                             and the Scytophycin B Derivatives vs. Fungal Pathogens                                    Size of Zone of Inhibition (mm)                                            Concen-  Saccharomyces                                                                             Neurospora                                                                            Candida                                          tration  pastorianus crassa  albicans                                Compound mg/mL    X52         X846    X657                                    ______________________________________                                        Scyto-   1        17          30      22                                      phycin C                                                                      Compound 1-7      19          28      20                                      Compound 1-8      --.sup.a    13      --                                      ______________________________________                                         --.sup.a = not active                                                    

                  TABLE 10                                                        ______________________________________                                        In Vitro Agar Diffusion Activity of Scytophycins A and B                      compared with Majusculamide C vs. Fungal Pathogens                                         Size of Zone of Inhibition (mm)                                                     Pythium  Rhizoctonia                                                                           Sclerotina                                          Conc.    ultimum  solani  homoeocarpa                               Compound  mg/mL    X751     X767    X768                                      ______________________________________                                        Sctophycin A                                                                            1.0      >30      30      >30                                                 0.1      >30      23       30                                                 0.01      20      13       20                                       Scytophycin B                                                                           1.0      30.sup.a ×20                                                                             >30                                                 0.1      30.sup.a >30     >30                                                 0.01      30      30      >30                                       Majuscul- 1.0      >30      20.sup.a                                                                               17                                       amide C   0.1       18      --      --                                                  0.01     10.sup.a --      --                                        ______________________________________                                         .sup.a Overgrowth                                                        

                  TABLE 11                                                        ______________________________________                                        In Vitro Agar Diffusion Activity of Scytophycins B, D and E                   vs. Fungal Pathogens                                                                       Size of Zone of Inhibition (mm)                                                     Pythium  Rhizoctonia                                                                           Sclerotina                                          Conc.    ultimum  solani  homoeocarpa                               Compound  mg/mL    X751     X767    X768                                      ______________________________________                                        Sctophycin B                                                                            1.0      30       46.sup.a                                                                              44                                                  0.33     30       40      40                                        Scytophycin D                                                                           1.0      27       30      26                                                  0.33     24       22      20                                        Scytophycin E                                                                           1.0      40       46.sup.a, 25                                                                          35                                                  0.33     30       33.sup.a, 15                                                                          33                                        ______________________________________                                         .sup.a Overgrowth                                                        

                  TABLE 12                                                        ______________________________________                                        In Vitro Agar Diffusion Activity of Scytophycin C and the                     Scytophycin B Derivatives vs. Fungal Pathogens                                             Size of Zone of Inhibition (mm)                                                     Pythium  Rhizoctonia                                                                           Sclerotina                                          Conc.    ultimum  solani  homoeocarpa                               Compound  mg/mL    X751     X767    X768                                      ______________________________________                                        Scytophycin C                                                                           0.5      12.sup.a 30      32                                        Compound  -7                                                                            1.0      12       32      32                                        Compound  -8                                                                            1.0      --.sup.b 15.sup.a                                                                              12                                        ______________________________________                                         .sup.a Overgrowth                                                             .sup.b --.sup.1 = not active                                             

The following examples are provided to illustrate this invention.

EXAMPLE 1 Production of Scytophycins A, B, C, D and E

Unialgal, non-axenic cultures of Scytonema pseudohofmanni ATCC 53141were grown in 25-L glass bottles containing an aqueous inorganic mediumhaving the following composition:

    ______________________________________                                        Ingredient         Amount                                                     ______________________________________                                        NaNO.sub.3         200       mg/L                                             NH.sub.4 Cl        10        mg/L                                             K.sub.2 HPO.sub.4.3H.sub.2 O                                                                     65        mg/L                                             MgSO.sub.4.7H.sub.2 O                                                                            50        mg/L                                             CaCl.sub.2.2H.sub.2 O                                                                            13        mg/L                                             3-(N-morpholino)-  627       mg/L                                             propanesulfonic                                                               acid                                                                          Minor elements     1         mL/L                                             solution.sup.a                                                                Trace elements     3/25 (0.12)                                                                             mL/L                                             solution.sup.b                                                                ______________________________________                                    

Prior to autoclaving, the pH of the complete medium is adjusted to 7with sodium hydroxide.

    ______________________________________                                        Ingredient            Amount                                                  ______________________________________                                        .sup.a Minor Elements Solution:                                               FeCl.sub.3 x6H.sub.2 O                                                                              0.54   g/L                                              Na.sub.2 EDTA         3.0    g/L                                              H.sub.3 BO.sub.3      0.62   g/L                                              MnCl.sub.2.4H.sub.2 O 1.4    g/L                                              ZnCl.sub.2            0.10   g/L                                              CoCl.sub.2.6H.sub.2 O 5      mg/L                                             CuCl.sub.2.2H.sub.2 O 34     mcg/L                                            ______________________________________                                        .sup.b Trace Elements Solution:                                                                (mg/10 L of 0.1 N H.sub.2 SO.sub.4)                          MoO.sub.3 (85%)  176.4                                                        NH.sub.4 VO.sub.3                                                                              229.6                                                        Cr.sub.2 K.sub.2 (SO.sub.4).sub.4.24H.sub.2 O                                                  960.2                                                        NiSO.sub.4.6H.sub.2 O                                                                          447.8                                                        Co(NO.sub.3).sub.2.6H.sub.2 O                                                                  493.8                                                        Na.sub.2 WO.sub.4.2H.sub.2 O 179.4                                            Al.sub.2 (SO.sub.4).sub.3                                                                      317.1                                                        As.sub.2 O.sub.3 66.1                                                         CdCl.sub.2       81.5                                                         SrSO.sub.4       104.9                                                        HgCl.sub.2       67.7                                                         PbCl.sub.2       67.1                                                         LiCl             305.5                                                        Rb.sub.2 SO.sub.4                                                                              78.1                                                         NaBr             64.4                                                         KI               65.4                                                         NaF              110.5                                                        Na.sub.2 SeO.sub.4                                                                             119.4                                                        Be(NO.sub.3).sub.2.3H.sub.2 O                                                                  1037.0                                                       ______________________________________                                    

The cultures were illuminated continuously at an incident intensity of330 microEinsteins m⁻² sec⁻¹ from banks of cool-white fluorescent tubesand were vigorously aerated with 0.5% carbon dioxide in air. Followingincubation at 24°±1° C. for periods ranging from 25 to 33 days, the algawas harvested by filtration or centrifugation at 5000×g in arefrigerated continuous flow centrifuge and then were freeze dried.Yields of the lyophilized algal cells were typically 0.3 to 0.4 g perliter of culture.

EXAMPLE 2 Isolation of Scytophycins A and B

Dried algal cells (23 g), produced as described in Example 1, werehomogenized in a Waring blender with one liter of ethanol-water (2:3)for ten minutes. The mixture was shaken overnight and then centrifugedat 12,000×g for twenty minutes. The supernate was concentrated underreduced pressure at 40° C. to a volume of 300 mL, and 100 mL of 95%ethanol was added to give 400 mL of an ethanol-water (1:3) solutionwhich was then subjected to sonication for two minutes to disperse afine floc.

This solution was passed through a 1-mL C-18 column (Sep-pak, MilliporeCorporation) in 10-mL portions at a flow rate of 5-10 mL/min. After thepassage of each 10-mL aliquot, the C-18 column was washed with 10 mL ofethanol-water (1:3). The crude scytophycins were eluted from the columnwith 15 mL of 50% ethanol-water. The column was then regenerated for thenext run by a reversed wash with 95% ethanol (to remove accumulatedprecipitate) followed by a normal wash with 5 mL of water. The combined50% ethanol-water effluent (600 mL) was concentrated in vacuo to removethe ethanol, and the aqueous concentrate (300 mL) was lyophilized.

The residual semisolid was dissolved in ethanol-water (7:3) andfiltered; the filtered solution (4.25 mL) was subjected to reverse-phaseHPLC in 200-μL portions on a Beckman 1-cm×25-cm column of ODS bonded to5-μm spherical silica-based packing, using 65% acetonitrile and 35%0.10M triethylamine and 0.122M acetic acid in water as the eluant. Usinga flow rate of 4 mL/min, a 200-μL aliquot could be introduced onto thecolumn every 10 minutes. Elution was monitored by ultraviolet absorptionat 300 nm. Two fractions having retention times of 6'20" (A) and 7'50"(B) were collected. Fractions A and B showed single peaks at 9'20" and12'46" when each one was rechromatographed on the same column, using57.5% CH₃ CN - 42.5% Et₃ N/HOAc buffer. Water (40 mL) was added to eachfraction, and the acetonitrile was evaporated under reduced pressure.The turbid aqueous mixture was then lyophilized twice to remove thevolatile buffer. Fraction A provided 14.3 mg of scytophycin A (0.062%),and fraction B yielded 34 mg of scytophycin B (0.15%).

EXAMPLE 3 Isolation of Scytophycins A, B, C, D and E

Freeze-dried algal cells (176 g), prepared as described in Example 1,were steeped in 6 L of dichloromethane/isopropanol (1:1) for 24 hours atroom temperature with stirring. The mixture was filtered, and theextract was evaporated to give a semi-solid residue. This material wasdissolved in 90% methanol (2 L), and the particulates were separated byfiltration. The filtrate was partitioned with hexane (2 L). The 90%methanol layer was adjusted in concentration to 65% methanol with waterand extracted with dichloromethane (2×2 L). The CH₂ Cl₂ layer wasevaporated to give a residue which was subjected to gel filtration onLH-20 Sephadex (83×9 cm) using hexane/dichloromethane (1:4) as theeluant.

The fractions eluting from 2100 mL to 2600 mL contained mostlyscytophycins B and C, whereas the fractions eluting from 2600 mL to 3100mL contained mostly scytophycin A with small amounts of scytophycins Dand E. Each set of fractions was dissolved in a minimum amount ofabsolute ethanol and applied to a 2-cm×0.9-cm column of BondElut C-18(Analytichem International, Harbor City, Calif.) which had beenpreviously equilibrated with 70% ethanol; the scytophycins wereseparated from most of the pigments by elution with 15 mL of 70%ethanol.

Final purification of the fraction containing scytophycins B and C wasachieved by reverse-phase HPLC on ODS-2 (Whatman M9 10/50 column) usingeither 70% acetonitrile/30% aqueous triethylammonium acetate buffer(0.1M triethylamine, 0.122M acetic acid) or 85% MeOH as the eluant.Scytophycin B (161 mg) eluted prior to scytophycin C (61 mg) on thiscolumn.

The fraction containing scytophycins A, D and E was further purified bynormal phase HPLC on silica using methanol/dichloromethane (3:97) as theeluant. After a small amount of scytophycin B was eluted, scytophycins D(6 mg), E (6 mg) and A (90 mg) were obtained.

EXAMPLE 4 7-O-Acetyl-scytophycin B

Scytophycin B was reacted with 1.2 equivalents of acetic anhydride, 1.2equivalents of triethylamine, and a catalytic amount of4-dimethylaminopyridine in methylene chloride at 25° C. for 5 hours togive 7-O-acetyl-scytophycin B.

EXAMPLE 5 Aldehyde 7 from Scytophycin B

Scytophycin B (18 mg) was dissolved in acetic acid (18 mL) and ethanol(18 mL). After being stirred at room temperature for 1 day, the reactionmixture was evaporated under reduced pressure. The residue was thenpassed through a Bond Elut column of C₁₈, eluting with 30 mL ofethanol:water (7:3) to give compound 7: UV λmax (MeOH): 264.5 nm(ε20,000); MS (FAB): m/z 783 (M+Na)⁺, 761 (M+H) and 729 (M-CH₃ OH+H)⁺.

EXAMPLE 6 Primary Alcohol 8 from Scytophycin B

Aldehyde 7, obtained as described in Example 5, was dissolved in ethanol(10 mL) and cooled at 0° C. To this solution, sodium borohydride (1 mg,1.1 equiv) in ethanol (0.5 mL) was added. The mixture was stirred at 0°C. for 30 minutes. After addition of acetone (2 mL), the reactionmixture was evaporated to a small volume (ca. 1 mL). Then brine andethyl acetate were added. This solution was extracted with ethyl acetateto give compound 8: UV λmax (MeOH): 263 nm (ε16,000); MS (FAB): m/z 785(M+Na)⁺ and 763 (M+H)⁺.

EXAMPLE 7 Di-p-Bromobenzoate of Compound 8

Compound 8, obtained as described in Example 6, and a catalytic amountof 4-dimethylaminopyridine were dissolved in dichloromethane (6 mL). Tothis solution, triethylamine (50 μL) and then p-bromobenzoyl chloride(30 mg) were added. After being stirred at room temperature for 1 day,the reaction mixture was poured onto a silica-gel column (0.8×7 cm)packed with dichloromethane. Elution with dichloromethane (20 mL) gaveunreacted acid chloride; then elution with dichloromethane/ethyl acetate(2:1) afforded crude product.

This product was further purified by silica-gel chromatography (0.8×7-cmcolumn), eluting with hexane/ethyl acetate (4:1) to give 9 mg of thedi-p-bromobenzoate of compound 8: mp: 105°-106° C. (MeOH-CH₂ Cl₂); ¹ HNMR (acetone-d₆): δ7.98 (m 4H, aromatic Hs), 7.74 (m 4H, aromatic Hs),7.49 (d, H-3), 6.01 (m, H-5), 5.85 (m, H-11), 5.78 (d, H-2), 5.68 (br d,H-10), 5.36 (br t, H-7), 5.25 (d, H-21), 4.31 (br d, H-9), 4.54 (dd,H-32), 4.27 (dd, H'-32), 3.82 (dd, H-15), 3.75 (dd, H-17), 3.42 (s,MeO), 3.31 (s, MeO), 3.29 (s, MeO), 3.23 (s, MeO), 2.70 and 2.60 (eachd, CH₂ -16), 1.88 (br s, Me-4), 1.12 (d, sec-Me), 1.09 (d, sec-Me), 0.92(d, sec-Me), 0.89 (d, sec-Me), and 0.79 (d, sec-Me).

EXAMPLE 8 Diacetate of Compound 8

Compound 8 (21 mg) was acetylated with acetic anhydride (0.5 mL) andpyridine (2 mL) at room temperature for 1 day. After addition ofmethanol (3 mL), the reaction mixture was evaporated and purified bysilica-gel column chromatography, eluting with hexane/ethyl acetate(2:1) to give 10 mg of the diacetate of compound 8: ¹ H NMR(acetone-d₆): δ7.45 (d, H-3), 5.92 (m, H-5), 5.83 (m, H-11), 5.76 (d,H-2), 5.64 (br d, H-10), 5.23 (d, H-21), 5.08 (t, H-7), 4.23 (d, H-9),4.20 (dd, H-32), 3.98 (dd, H-32), 3.79 (dd, H-15), 3.73 (dd, H-17), 3.38(s, MeO), 3.27 (s, MeO), 3.24 (s, MeO), 3.20 (s, MeO), 2.67 and 2.57(each d, CH₂ O), 1.85 (br d, Me-4), 1.07 (d, sec-Me), 1.07 (d, sec-Me),0.89 (d, sec-Me), 0.89 (d, sec-Me) and 0.82 (d, sec-Me).

EXAMPLE 9 7-O-(p-Bromobenzoyl)scytophycin C

Scytophycin C (15 mg) and 4-dimethylaminopyridine (a catalytic amount)were dissolved in dichloromethane (3 mL). To this solution,triethylamine (100 μL) and then p-bromobenzoyl chloride (50 mg) wereadded. After being stirred at room temperature for 8 hours, the mixturewas poured onto a silica-gel column (0.7×8 cm). Elution withdichloromethane (20 mL) gave unreacted acid chloride; then elution withethyl acetate/dichloromethane (1:1) afforded crude products.Purification by reverse-phase HPLC (Whatman ODS 2), eluting with MeOH/H₂O (94:6), afforded 7-O-(p-bromobenzoyl)scytophycin C: UV λmax (MeOH):252.5 nm (ε31,600); ¹ H NMR (acetone-d₆): δ8.35 (s, H-34), 8.11 (s, H-34of the minor conformer), 7.98 (d, aromatic Hs), 7.74 (d, aromatic Hs),7.67 (d, H-3), 6.78 (d, H-32), 7.10 (d, H-32 of the minor conformer),6.18 (t, H-5), 5.83 (d, H-2), 5.82 (m, H-11), 5.75 (d, H-10), 5.45 (m,H-7), 5.18 (d, H-21), 5.11 (dd, H-31), 4.35 (d, H-9), 4.14 (d, OH-23),3.71 (br d, H-15), 3.54 (t, H-17), 3.35 (s, MeO), 3.34 (s, MeO), 3.31(s, MeO), 3.21 (s, MeO), 3.00 (s, Me-N33), 3.12 (s, Me-N33 of the minorconformer), 1.89 (br s, Me-4), 1.16 (d, sec-Me), 0.99 (d, sec-Me), 0.95(d, sec-Me), 0.94 (d, sec-Me), 0.88 (d, sec-Me) and 0.86 (d, sec-Me).

EXAMPLE 10 Scytophycin B Antifungal Composition

An antifungal composition containing scytophycin B can be prepared asfollows:

Dissolve scytophycin B (10 mg) in 1 mL of acetone:ethanol (1:1) to which5 to 10% of a surfactant such as Tween 20 has been added. Add water (99mL) to result in a final concentration of 100 ppm of scytophycin B, 1%solvent and 0.05 to 0.1% surfactant.

This antifungal solution can be sprayed on plants or soil to inhibitfungal pathogens.

We claim:
 1. A biologically purified culture of Scytohema pseudohofmanniATCC 53141 or a scytophycin-producing mutant of said culture.
 2. Theculture of claim 1 which is S. pseudohofmanni ATCC 53141.